Project Summary The goal of this proposal is to determine how the FoxO1 transcription factor contributes to human liver development as a mediator of gene expression and metabolism in the differentiating hepatocyte. This knowledge will contribute to a more complete understanding of the mechanisms underlying hepatocyte differentiation and function necessary for tackling pervasive human metabolic disorders, as well as improving hepatocyte derivation from pluripotent stem cells. FoxO1 is classified as a ?pioneer? or initial chromatin binding transcription factor due to its unique ability to open silent compacted chromatin and perturb underlying histone:DNA contacts to promote recruitment of additional regulatory factors. We have used a novel human induced pluripotent stem cell (hiPSC) hepatocyte differentiation system to show, for the first time, that stage- specific disruption of FoxO1 chromatin binding prevents the establishment of hepatic progenitors within the definitive endoderm and severely curtails hepatic specification, the first two stages of liver development. This discovery raises the exciting possibility that FoxO1 obligatorily regulates the network of genes that is required to specify hepatic fate in the human embryo. Additionally, we've show that, in human hepatocytes, interdependent chromatin binding by FoxO1 and another liver-enriched pioneer factor, FoxA, plays an essential role in maintaining an active chromatin environment as well as the binding of transcription factors that induce insulin-regulated genes. Taken together with a recent study showing that FoxO1 and FoxA co-target multiple genes linked to metabolic pathways for glucose, lipids, cholesterol, and bile acids, this finding points to interdependent FoxO1/FoxA binding as a general regulatory mechanism enabling the creation and maintenance of active chromatin states in response to extracellular cues for a broad array of hepatic metabolic processes. Based on this evidence, we hypothesize that FoxO1 uses its diverse chromatin binding and remodeling capabilities to play two distinct roles in human liver development: (1) participation in key developmental pathways as a transcriptional regulator of target genes required for hepatocyte specification and differentiation followed by (2) cooperation with FoxA factors to activate and modulate expression of metabolic genes essential for the mature hepatocyte. We propose to investigate this hypothesis by identifying the essential gene regulatory networks induced by FoxO1 that are responsible for specification of human hepatic fate and determining how interdependent FoxO1/FoxA binding and transcription regulatory factor recruitment impacts their activation of essential hepatic metabolic functions. Subversion of the corresponding gene regulatory events is a likely contributor to metabolic derangements and hepatic disease, making it vital that we uncover the key mechanisms and players.